Anesthetic gasses are administered to thousands of patients in operating rooms across the United States each day. These agents are generally well tolerated but, in addition to the central nervous system depression they produce, the anesthetic gasses, or volatile anesthetics (VAs), have a number of potentially harmful effects. One of the most notable of these is a powerful smooth muscle relaxing action which can contributes to significant, and potentially dangerous, decreases in blood pressure. The goal of the research described in this proposal is to determine the molecular mechanism(s) responsible for the vascular smooth muscle relaxing action of VAs. Contraction in smooth muscle is initiated by increases in intracellular Ca+2 concentration ([Ca2+]i), which activate myosin light chain (MLC) kinase, thereby resulting in MLC phosphorylation and initiation of contraction. Utilizing techniques developed in this laboratory for measuring contraction, [Ca2+]i, and MLC phosphorylation in smooth muscle from small resistance arteries, studies to date on this project indicate that vascular smooth relaxation by the VA halothane occurs by a novel mechanism whereby MLC phosphorylation is inhibited in the absence of any effect on [Ca2+]i. This suggests that halothane acts either by inhibiting MLC kinase (MLCK) or activating MLC phosphatase (MLCP), independent of changes in [Ca2+]i. Ongoing studies described in this proposal will validate the importance of this mechanism for other commonly used Vas, determine whether the effect primarily involves inhibition of MLCK or activation of MLCP, and explore the oinvolvement of known intracellular modulators of MLCK and MLCP activity. In addition, using the minimally disruptive permeabilizing agent staphylococcal a-toxin, we have successfully reconstituted halothane-induced relaxation, previously only observed in intact smooth muscle, in a membrane permeabilized preparation. This latter model provides access of specific peptide antagonists to the intracellular milieu, and will be utilized to further explore the involvement of defined modulators of MLCK and MLCP in VA induced relaxation. Ultimately, these combined approaches will lead to identification of the precise molecular mechanism(s) involved in the vascular smooth muscle relaxing action of VAs.